Elife 92393 v1

Research Article
Antipsychotic-induced epigenomic
reorganization in frontal cortex of
individuals with schizophrenia
Bohan Zhu1†, Richard I Ainsworth2†‡, Zengmiao Wang2, Zhengzhi Liu3,
Salvador Sierra4§, Chengyu Deng1, Luis F Callado5, J Javier Meana5, Wei Wang2,6*,
Chang Lu1*, Javier González-Maeso4*
1
Department of Chemical Engineering, Virginia Tech, Blacksburg, United States;
2
Department of Chemistry and Biochemistry, University of California, San Diego,
*For correspondence: La Jolla, United States; 3Department of Biomedical Engineering and Mechanics,
wei-wang@ucsd.edu (WW); Virginia Tech, Blacksburg, United States; 4Department of Physiology and Biophysics,
changlu@vt.edu (CL); Virginia Commonwealth University School of Medicine, Richmond, United States;
javier.maeso@vcuhealth.org 5
Department of Pharmacology, University of the Basque Country UPV/EHU,
(JG-M)
CIBERSAM, Biocruces Health Research Institute, Bizkaia, Spain; 6Department of
†
These authors contributed Cellular and Molecular Medicine, University of California, San Diego, La Jolla, United
equally to this work
States
Present address: ‡Department
of Medicine, Kao Autoimmunity
Institute, Cedars-Sinai
Medical Center, Los Angeles, Abstract Genome-wide association studies have revealed >270 loci associated with schizo-
United States; §Department phrenia risk, yet these genetic factors do not seem to be sufficient to fully explain the molecular
of Neurology, University of
determinants behind this psychiatric condition. Epigenetic marks such as post-translational histone
Michigan, Ann Arbor, Ann Arbor,
modifications remain largely plastic during development and adulthood, allowing a dynamic impact
United States
of environmental factors, including antipsychotic medications, on access to genes and regulatory
Competing interest: See page elements. However, few studies so far have profiled cell-specific genome-wide histone modifi-
22 cations in postmortem brain samples from schizophrenia subjects, or the effect of antipsychotic
Funding: See page 23 treatment on such epigenetic marks. Here, we conducted ChIP-seq analyses focusing on histone
Preprint posted
marks indicative of active enhancers (H3K27ac) and active promoters (H3K4me3), alongside RNA-
29 August 2023 seq, using frontal cortex samples from antipsychotic-free (AF) and antipsychotic-treated (AT) indi-
Sent for Review viduals with schizophrenia, as well as individually matched controls (n=58). Schizophrenia subjects
10 September 2023 exhibited thousands of neuronal and non-neuronal epigenetic differences at regions that included
Reviewed preprint posted several susceptibility genetic loci, such as NRG1, DISC1, and DRD3. By analyzing the AF and AT
07 December 2023 cohorts separately, we identified schizophrenia-associated alterations in specific transcription
Reviewed preprint revised factors, their regulatees, and epigenomic and transcriptomic features that were reversed by anti-
27 March 2024
psychotic treatment; as well as those that represented a consequence of antipsychotic medication
Version of Record published
rather than a hallmark of schizophrenia in postmortem human brain samples. Notably, we also
22 April 2024
found that the effect of age on epigenomic landscapes was more pronounced in frontal cortex of
Reviewing Editor: Charlotte AT-schizophrenics, as compared to AF-schizophrenics and controls. Together, these data provide
Cecil, Erasmus MC, Netherlands important evidence of epigenetic alterations in the frontal cortex of individuals with schizophrenia,
‍ ‍Copyright Zhu, Ainsworth and remark for the first time on the impact of age and antipsychotic treatment on chromatin
et al. This article is distributed organization.
under the terms of the Creative
Commons Attribution License,
which permits unrestricted use
and redistribution provided that eLife assessment
the original author and source The study by Zhu et al. provides important insights into cell-specific genome-wide histone modifi-
are credited. cations in the frontal cortex of individuals with schizophrenia, as well as shedding light on the role of
Zhu, Ainsworth et al. eLife 2023;12:RP92393. DOI: https://doi.org/10.7554/eLife.92393 1 of 29

Research article Genetics and Genomics | Neuroscience
age and antipsychotic treatment in these associations. The evidence supporting the conclusions is
solid.
Introduction
Schizophrenia has traditionally been viewed as a genetic disorder, with heritability rates estimated
at ~73% (Sawa and Snyder, 2002; Freedman, 2003). However, previous genome-wide association
studies (GWAS) clearly showed a relatively low number of genetic regions associated with schizo-
phrenia risk – these include 108 loci in the first study (Schizophrenia Working Group of the Psychi-
atric Genomics Consortium, 2014) that have been expanded to over 270 regions (Farrell et al.,
2015; Trubetskoy et al., 2022). Most of these genetic variants were located in non-coding regions
and hence, with a few exceptions there is little evidence supporting that coding variants contribute
to schizophrenia risk, which also suggests that genetic factors do not seem to be sufficient to fully
explain the molecular causes underlying this severe psychiatric condition.
Twin studies have provided evidence that environmental factors contribute to schizophrenia
susceptibility. Thus, it had originally been suggested that monozygotic twins, whose DNA sequences
are approximately 100% identical, have a concordance for schizophrenia of nearly 50% (Cardno and
Gottesman, 2000; Hallmayer et al., 2011), yet recent research has revealed a probandwise concor-
dance rate of 33% in monozygotic twins and 7% in dizygotic twins (Hilker et al., 2018). While these
findings underscore the substantial influence of genetic factors on the etiology of schizophrenia,
they also advocate for a significant involvement of environmental events in the intricate develop-
ment of this complex disorder (Li et al., 2021). This concept is further supported by epidemiological
studies suggesting that prenatal environmental insults, such as maternal infection (Brown et al., 2004;
Yudofsky, 2009) and severe adverse life events (Malaspina et al., 2008), increase the risk of schizo-
phrenia in the offspring.
Gene expression is regulated by the ability of the transcriptional machinery to access DNA, which
is tightly packed into chromatin. The status of chromatin organization depends on epigenetic factors,
such as DNA methylation and histone modifications that primarily occur on amino-terminal tails (Gräff
and Tsai, 2013; Onuchic et al., 2018; Bastle and Maze, 2019). Hence, these epigenetic mechanisms
lead to stable changes in gene expression that are mediated via altered chromatin structure without
modification of DNA sequence, and remain largely plastic throughout all periods of brain develop-
ment and aging. It is then tempting to speculate that epigenetic mechanisms mediate, at least in part,
the effects of environmental factors on central nervous system (CNS) gene activity, and are, therefore,
potentially involved in the pathophysiology of schizophrenia and other mental illnesses.
Supporting this concept, previous studies reported alterations in chromatin structure and acces-
sibility in tissue samples from schizophrenic subjects and controls. Most of these previous reports,
however, were focused on DNA methylation differences in peripheral blood (Aberg et al., 2014)
and brain (Jaffe et al., 2016; Mendizabal et al., 2019). Using the assay for transposase-accessible
chromatin sequencing (ATAC-seq) in bulk tissue homogenates of postmortem frontal cortex samples,
only a few differences in chromatin accessibility were observed between schizophrenia subjects and
controls, in contrast to thousands of age-related differential accessible chromatin regions (Bryois et al.,
2018). Histone modifications, including histone H3 acetylation of lysine 27 (H3K27ac) and histone H3
trimethylation of lysine 4 (H3K4me3) are critically involved in epigenomic regulations; H3K27ac marks
active enhancers (Creyghton et al., 2010), whereas H3K4me3 marks active promoters (Bernstein
et al., 2005). Enhancers are highly dynamic cis-regulatory elements with known involvement in neuro-
developmental processes (Won et al., 2019), and the dynamics of promoters are also significantly
connected with the genetic risk of certain psychiatric conditions (Dincer et al., 2015). However, very
few studies have been conducted about potential cell-type-specific genome-wide variations in cova-
lent histone modifications in postmortem human brain samples of individuals with schizophrenia.
As an example, recent work combined fluorescence-activated cell sorting (FACS) of neuronal and
non-neuronal cell nuclei with chromatin immunoprecipitation sequencing (ChIP-seq) assays in two brain
regions (prefrontal cortex and anterior cingulate cortex) from postmortem brain samples of subjects
without any known neurological or psychiatric disease (Girdhar et al., 2018). Besides the identifica-
tion of cell and region-specific histone modification landscapes in this cohort of control subjects, these
findings also compared their datasets with previous GWAS of individuals with psychiatric conditions,

reporting that strong specific enrichments occurred with schizophrenia and weaker associations with
depression in both H3K27ac and H3K4me3 peaks. This correlation was almost exclusively observed
in neuronal chromatin, but not in non-neuronal cell nuclei. More recent investigation conducting
H3K27ac and H3K4me3 ChIP-seq assays in cortical neurons from schizophrenia subjects and controls
has identified rare specific epigenetic variations for a set of non-coding RNA genes (Gusev et al.,
2019) and chromatin domain alterations (Girdhar et al., 2022) that may contribute to the patho-
genesis of schizophrenia. However, these previous epigenomic studies in postmortem human brain
samples did not address the potential effect of previous exposure to antipsychotics on the regulation
of chromatin state. This is particularly relevant considering that repeated administration of antipsy-
chotic medications leads to epigenetic modifications at selected gene regions in mouse (de la Fuente
Revenga et al., 2018; Ma et al., 2018) and postmortem human brain (Kurita et al., 2012; Ibi et al.,
2017) samples. Similarly, whether such type of schizophrenia-associated epigenomic changes are
observable in non-neuronal frontal cortex nuclei remains unexplored; even though there is evidence
that alterations in the glia may contribute to major psychiatric disorders (Liu et al., 2022).
Combining MOWChIP-seq (Cao et al., 2015a; Zhu et al., 2019) and Smart-seq2 (Picelli et al.,
2013) for low-input profiling of H3K27ac and H3K4me3 histone modifications and transcriptomes,
respectively, here we present the first dataset with cell type-specific epigenomic and transcriptomic
landscapes in postmortem frontal cortex samples from two cohorts of schizophrenics either previously
treated or not with antipsychotic medications and control subjects individually matched by sex and
age. Importantly, our analyses allow the identification of transcription factors (TFs), their regulatees,
and genes that may be involved in either the therapeutic effects of antipsychotics or the cause of
undesired antipsychotic-induced epigenomic aberrations.
Results
Quality assessment for ChIP-seq and RNA-seq datasets in frontal
cortex from postmortem human brain samples
Frontal cortex is a brain region involved in processes affected in schizophrenia patients, such as
perception, cognition, and sensorimotor gating (Andreasen et al., 1994). We selected bilateral frontal
cortex (Brodmann area 9) gray matter from 58 brain samples (29 schizophrenia and 29 controls).
Control subjects were individually matched based on sex and age, and to a lower degree on post-
mortem delay (or PMD – time between death and tissue sample collection) (Supplementary files 1
and 2). Nuclei were FACS-sorted using an anti-NeuN antibody as a marker of neuronal nuclei, and
NeuN-positive (NeuN+) and NeuN-negative (NeuN-) nuclei (approximately 60,000 nuclei per NeuN+ or
NeuN- sample) were collected for ChIP-seq (10,000 nuclei per library) and RNA-seq (6000 nuclei per
library) (Figure 1A). After library preparation and sequencing, our MOWChIP-seq technology gener-
ated high-quality ChIP-seq data with average unique reads of ~11 million and ~14 million on histone
modifications H3K27ac and H3K4me3, respectively (Figure 1—figure supplement 1A; and Supple-
mentary file 3). These yields were comparable to those in our previous studies using mouse frontal
cortex and mammalian tissue culture samples (Zhu et al., 2019; de la Fuente Revenga et al., 2021).
We generated saturation curves to validate that our sequencing depth is sufficient, and that a further
increase in the sequencing depth would not lead to significantly more called peaks (Figure 1—figure
supplement 2). MOWChIP-seq datasets have very low background noise (de la Fuente Revenga
et al., 2021) with the fraction of reads in called peaks (FRiP) average at 17.35% and 27.59% for our
H3K27ac and H3K4me3 profiling, respectively (Figure 1—figure supplement 1B; and Supplemen-
tary file 3). The PCR bottleneck coefficient (PBC) was calculated to measure library complexity (0.90
and 0.92 for H3K27ac and H3K4me3, respectively), which indicates that most of our ChIP-seq datasets
have no or mild bottlenecking (Figure 1—figure supplement 1C; and Supplementary file 3).
Using Phantompeakqualtools (Marinov et al., 2014), we calculated the normalized strand cross-
correlation (NSC) and relative strand cross- correlation (RSC) to demonstrate the enrichment of
sequencing reads around the histone modification sites (Figure 1—figure supplement 1D and E; and
Supplementary file 3). The average NSC was 1.15 and 1.23 for H3K27ac and H3K4me3, respectively;
and the average RSC was 3.75 and 2.09 for H3K27ac and H3K4me3, respectively. These NSC and RSC
values were higher than the recommended thresholds of 1.05 and 1.0, respectively. We also compared
our GC metrics with those of previously published ChIP-seq data in postmortem human frontal cortex

Figure 1. Overview of the multi-omics protocol for analyzing frontal cortex of schizophrenia subjects and controls.
(A) Overview of the experimental design starting from postmortem human frontal cortex samples to generate
cell type-specific H3K27ac, H3K4me3, and RNA profiles. (B) Heatmap of the expression of neuronal and glial cell
markers across all NeuN+ and NeuN- frontal cortex samples from 29 control subjects and 29 schizophrenia subjects.
Figure 1 continued on next page

Figure 1 continued
The online version of this article includes the following figure supplement(s) for figure 1:
Figure supplement 1. Distribution of six quality control metrics for ChIP-seq data on H3K4me3 and H3K27ac in
NeuN-positive (NeuN+) and NeuN-negative (NeuN-) nuclei from schizophrenia and control groups, respectively.
Figure supplement 2. Saturation curves on the relationship between the number of sequencing reads and the
number of identified peaks with our ChIP-seq data on H3K4me3 and H3K27ac.
Figure supplement 3. Distribution of four quality control metrics for RNA-seq data in NeuN-positive (NeuN+) and
NeuN-negative (NeuN-) nuclei from schizophrenia and control groups.
Figure supplement 4. Representative results from fluorescence-activated cell sorting (FACS) sorting
demonstrating the separation of neuronal (NeuN+) and non-neuronal (NeuN-) nuclei using fluorescence-labeled
anti-NeuN antibody in postmortem human frontal cortex samples.
Figure supplement 5. Expression of neuronal (NeuN+) and non-neuronal (NeuN-) representative marker genes.
Figure supplement 6. Venn diagrams of the overlap between the identified peaks from our ChIP-seq study
(green) and previous datasets (Girdhar et al., 2022) (cyan).
Figure supplement 7. Visualization of peak-wise or gene-wise means and variances of ChIP-seq and RNA-seq
data, respectively, by voom plots.
samples (Girdhar et al., 2018). This previous study had an average NSC of 1.33 and 2.81, and RSC of
1.18 and 1.06 for H3K27ac and H3K4me3, respectively. The average Pearson’s correlations between
replicates were 0.923 and 0.971 for H3K27ac and H3K4me3, respectively, which compare well with
those of ENCODE data (ENCODE Project Consortium, 2012). Our RNA-seq datasets had an average
of ~6.8 million uniquely mapped reads, and the average mapping rate was 82.5% (Figure 1—figure
supplement 3; and Supplementary file 4), within the recommended range of 70–90% (Conesa et al.,
2016). Our average GC content was 40.73% and exon percentage was 36.52%.
Using all frontal cortex samples from the 29 schizophrenia subjects and 29 controls, we analyzed the
expression of selected neuronal and non-neuronal marker genes. Highly significant (median p-value
= 6×10–7) pair-wise differences in molecular marker expression were observed for all markers ranging
from mature, functional, and synaptic neuron markers to astrocyte, oligodendrocyte, and microglial
markers (Figure 1B; Figure 1—figure supplements 4 and 5; Supplementary file 5) – confirming
neuronal and non-neuronal cell-type identities in the NeuN+ and NeuN- nuclei samples, respectively.
Consequently, it can be concluded that MOWChIP-seq technology offers data quality compa-
rable to that of state-of-the-art standard reference epigenomes while allowing histone modification
profiling using small and highly purified populations of neuronal and non-neuronal nuclei from post-
mortem human frontal cortex samples.
Epigenome and transcriptome profiling in neuronal and non-neuronal

nuclei from frontal cortex of schizophrenia subjects and controls
To gain insight into the cell type-specific epigenomic changes associated with schizophrenia, we
profiled histone marks H3K27ac and H3K4me3 and transcriptomes in NeuN+ and NeuN- nuclei from
the frontal cortex of schizophrenia subjects and controls. MOWChIP-seq data were mapped to the
reference genome (Grch38), and significant peaks were called using MACS2 (Figure 1—figure supple-
ment 1F). We then performed the overlap analysis to identify consensus peak sets for H3K27ac and
H3K4me3 in NeuN+ and NeuN- nuclei (see Methods section). We identified 107,938 consensus peaks
covering ~135 Mb (4.22% of the genome) for H3K27ac in NeuN+ nuclei, 71,490 consensus peaks
covering ~101 Mb (3.18% of the genome) for H3K27ac in NeuN- nuclei, 13,3137 consensus peaks
covering ~164 Mb (5.12% of the genome) for H3K4me3 in NeuN+ nuclei, and 100,745 consensus
peaks covering ~142 Mb (4.43% of the genome) for H3K4me3 in NeuN- nuclei. This peak distribu-
tion was consistent with previous H3K27ac and H3K4me3 NeuN+ ChIP-seq studies in postmortem
human frontal cortex tissue samples (Girdhar et al., 2022; Figure 1—figure supplement 6). Voom
plots using raw binding affinity matrix in ChIP-seq and RNA-seq datasets validate that low-enriched/
expressed peaks/genes were filtered before downstream analysis (Figure 1—figure supplement 7).
This is further corroborated with the smoothly decreasing curves fitted to the square root of residual
standard deviation by average expression in all cases (Figure 1—figure supplement 7).

We then investigated the differences in histone modification profiles and gene activity between the
schizophrenia and control cohorts. We regressed demographic (sex, age at death, PMD, antemortem
diagnosis) and technical (align rate, unique rate, FRiP, NSC, RSC, the number of identified peaks,
and PBC) covariates (Sun et al., 2016). We defined differential H3K27ac peaks that have no overlap
regions with promoters as differential enhancers. Our analysis revealed 2,301 differential enhancers,
262 differential promoters, and 802 differentially expressed genes (DEGs) in NeuN+ nuclei between
schizophrenia subjects and controls, while 2,657 differential enhancers, 360 differential promoters, and
1,043 DEGs were discovered in NeuN- nuclei (Figure 2A; Supplementary file 6). We next leveraged
the previously identified promoter-anchored chromatin loops in NeuN+ and NeuN- nuclei to identify
how differential enhancers are linked with genes (Hu et al., 2021). We successfully associated 639 and
714 cell-type-specific enhancers to 328 and 395 genes via enhancer-promoter interactions in NeuN+
and NeuN- nuclei, respectively. The rest of the enhancers were associated with their nearest genes.
We discovered that the schizophrenia group had varied H3K27ac/H3K4me3 levels and RNA-seq reads
compared to controls at various loci involved in processes related to synaptic plasticity and cogni-
tive processes or previously associated with schizophrenia risk (Farrell et al., 2015) – these included
enhancer region of NRG1 (Neuregulin 1) in NeuN+ nuclei, enhancer region of GRM3 (Metabotropic
glutamate 3 receptor) in NeuN- nuclei, promoter region of DRD3 (Dopamine D3 receptor) in NeuN+
nuclei, promoter region of DISC1 in NeuN- nuclei, CDK5 (Cyclin-dependent kinase 5) mRNA in NeuN+
nuclei, and GRIN2A (Glutamate ionotropic receptor NMDA type subunit 2 A) mRNA in NeuN- nuclei
(Figure 2B and C; Supplementary file 6). The accuracy of H3K27ac and H3K4me3 differential peak
calling between schizophrenia subjects and controls identified by MOWChIP-seq was validated by
independent ChIP-qPCR analysis of selected loci (Figure 2—figure supplement 1).
We also constructed QQ plots to examine the validity of our differential analysis (Figure 2—figure
supplement 2). The lambda values of our H3K4me3 ChIP-seq datasets were lower than 1 (i.e. 0.43 and
0.36 for NeuN+ and NeuN- nuclei, respectively), suggesting that there are fewer differential promoters
than in the normal distribution. This matches previous findings on postmortem human brain samples
(Girdhar et al., 2018).
To further explore the association between differential epigenetic modifications and genetic loci
previously associated with schizophrenia risk (Trubetskoy et al., 2022), we examined the overlap
of differential enhancer and promoter peaks with genetic variants using linkage disequilibrium (LD)
score regression (Finucane et al., 2015). Various other brain and non-brain-related traits were also
considered for comparison (Finucane et al., 2018). Schizophrenia was the most significantly enriched
trait for all the differential enhancer and promoter regions in NeuN+ and NeuN- fractions (Supplemen-
tary file 7). Additionally, the level of enrichment was higher in differential enhancers as compared to
promoters, and the highest in differential enhancers of NeuN+ nuclei (Supplementary file 7).
To assess agreement with the literature, we compared the DEGs identified in our study with a
previous single-nucleus RNA sequencing (snRNA-seq) study in the postmortem prefrontal cortex of
schizophrenics and controls (Ruzicka et al., 2020). Importantly, 236 out of our 802 DEGs (p-value =
1.96×10–11) in NeuN+ nuclei, and 63 out of our 1,043 DEGs (p-value = 4.18×10–6) in NeuN- nuclei were
also identified in this previous single-cell dissection work. In NeuN+ nuclei, several genes encoding
metabotropic glutamate receptors (GRM3, GRM5) that are directly associated with schizophrenia risk
(Maj et al., 2016) were found differentially expressed in both studies (Supplementary file 8). We
also identified some novel genes, including LRRTM3, which regulates excitatory synapse develop-
ment (Um et al., 2016), and POU3F2, which is viewed as a key regulator of gene expression in a
schizophrenia-associated gene co-expression module (Chen et al., 2018; Supplementary file 8).
We also overlapped genes identified from differential enhancers/promoters with DEGs from RNA-
seq (Figure 2D; Supplementary file 9). We found that 66 (p-value = 9×10–3) and 148 (p-value =
1.7×10–2) genes were identified in both the DEGs from RNA-seq and differential enhancers/promoters
associated genes in NeuN+ and NeuN- nuclei, respectively. Among these, several schizophrenia-
associated genes were also detected, including NTNG2, which is known to be involved in neurodevel-
opmental disorders (Dias et al., 2019) and GRIN3A, a gene that encodes NMDA receptor subunits in
neuronal nuclei (Yu et al., 2018).
For integrative analysis of these diverse epigenomic and transcriptomic data, we next employed an
unbiased method called EpiSig (Ai et al., 2018), which combines the epigenomic and transcriptomic
dataset into a single analysis to cluster regions with similar epigenomic profiles across all the NeuN+

Figure 2. Comparison of epigenomic and transcriptomic landscapes in the frontal cortex of schizophrenia subjects and controls. (A) Differential
enhancer/promoter peaks and differentially expressed genes (DEGs) obtained by comparing schizophrenia (n=29) and controls (n=29). The
differential peaks or DEGs were identified using FDR <0.05. (B) Exemplar genomic track view of H3K27ac, H3K4me3, and RNA signals for matched
AF-schizophrenia/control and AT-schizophrenia/control pairs in NeuN-positive (NeuN+) and NeuN-negative (NeuN-) cells. 50 Mb region displayed:

Figure 2 continued
chr1:68,000,000–118,000,000 (GRCh38). (C) Volcano plots showing genes associated with differential enhancer and promoter peaks and DEGs.
Candidate genes for schizophrenia or genes involved in significant GO terms are labeled. The horizontal lines indicate FDR of 0.05. (D) Venn diagrams
on the relationship among genes associated with differential enhancer or promoter peaks and DEGs.
Figure supplement 1. qPCR validation of selected differential ChIP-seq peaks in NeuN+ fraction for H3K27ac (a-c) and H3K4me3 (d-f).
Figure supplement 2. Q-Q plots of the corrected p-value from differential peaks at enhancer/promoter regions and differentially expressed genes.
and NeuN- nuclei samples. 85,462 signal-enriched regions were grouped into 814 epigenomic clus-
ters covering 14.53% of the genome. These clusters were further combined into 6 groups (sections)
using the K-means method (Figure 3A and B; Supplementary files 10 and 11). Section I had high
coverage in the gene annotations for intron (35%) and intergenic regions (29%) indicating inactive
regions. It was also enriched in chromosome X compared to other sections. Section II was anno-
tated as enhancers that are active in NeuN+ nuclei but suppressed in NeuN- nuclei. A hypergeometric
test identified clusters that were significantly enriched in schizophrenia vs control differential histone
marks and differentially expressed genes (Figure 3A and B; Supplementary files 10 and 11). The
top five Section II clusters had schizophrenia-positive association (i.e. activity and expression schizo-
phrenia >controls) in genes enriched in GO terms ‘Trans-synaptic signaling’ (FDR 4.66×10–2). Section III
was highly enriched in enhancers (average of 21% of all regions in each cluster), and had low coverage
in intergenic regions (12%), which is likely associated with active enhancers for both NeuN+ and NeuN-
given the high signal strength for H3K27ac. Top differentially enriched cluster genes, enriched in the
GO term ‘Amyloid fibril formation’ (FDR 5.99×10–2), were found to be negatively associated with
schizophrenia in NeuN+ nuclei, whereas genes enriched in the GO term ‘Neuron projection develop-
ment’ (FDR 3.19×10–2) were positively associated with schizophrenia in NeuN- nuclei (Figure 3A and
B; Supplementary files 10 and 11). Section IV also had high coverage of enhancers (Figure 3A and B;
Supplementary files 10 and 11). However, it had the highest average promoter content with 16% of
all gene annotations being promoter regions, further supported by CpG islands showing the highest
proportion (20%) in this section; indicating active promoters for both NeuN+ and NeuN- nuclei. Both
cell types showed enrichment in respiratory electron transport genes that were negatively associated
with schizophrenia (Figure 3A and B; Supplementary files 10 and 11). As for the average differ-
ential signals across sections, a great variance was observed. For example, the signal of H3K4me3
was higher in schizophrenia subjects compared to controls for NeuN+ nuclei in section V, while it was
lower in schizophrenia subjects for NeuN- samples (Figure 3A and B; Supplementary files 10 and
11). Finally, Section VI was annotated as enhancers that were active in NeuN- nuclei but repressed in
NeuN+ nuclei (Figure 3A and B; Supplementary files 10 and 11).
Transcriptional regulatory processes proceed as a hierarchy of orchestrated events that ultimately
modulate the expression of downstream target genes. Using the recently developed Taiji algorithm
(Zhang et al., 2019), which allows access to information pertaining to transcriptional cascades
deriving from upstream drivers through specific pathway mechanisms to downstream effects, we inte-
grated epigenomic and transcriptomic data to construct 116 individual transcriptional networks in
neuronal and glial nuclei from schizophrenia subjects and controls. We identified active promoters
and enhancers using H3K27ac and then predicted TF binding sites by scanning 1,165 TF motifs linking
putative TF binding sites to their targets using EpiTensor (Zhu et al., 2016), an unsupervised method
to predict enhancer-promoter associations. TFs were subsequently ranked according to regulatory
importance using the Personalized PageRank (PPR) algorithm for each unique network topology (Yu
et al., 2017). Using the differentially expressed TFs (schizophrenia vs controls FDR <0.05), TFs were
ranked by absolute change in schizophrenia vs control PPR score (Figure 4A – top 10 TFs for each
cell type; Supplementary file 12). Of the top 10 TFs of NeuN+ nuclei, all were found to be cell-type
specific. Using the top four TFs, we identified 207 regulatees the were regulated by three or more
TFs and found they were involved in processes such as ‘Neurexins and neuroligins’ (FDR 2.18×10–7)
and ‘Protein-protein interactions at synapses’ (FDR 1.22×10–6) (Figure 4B; Supplementary file 12).
Furthermore, all top 10 TFs of NeuN- nuclei were cell-type specific TFs and the regulatees of the top
four TFs were enriched in signaling pathways including ‘RAF/MAP kinase cascade’ (FDR 2.80×10–2)
and ‘RHO GTPase cycle’ (FDR 3.07×10–2) (Figure 4C; Supplementary file 12).

Figure 3. Genome-wide multidimensional clusters in the frontal cortex of schizophrenia subjects and controls.
(A) Integrative analysis using EpiSig. 814 EpiSig clusters across 348 genome-wide sequencing datasets were
grouped into 6 sections. The heatmap shows the signal in each EpiSig cluster (row: EpiSig cluster; column: marker).
(B) For each EpiSig cluster, from left to right, the heatmaps are: the region percentage in each chromosome;
the genomic annotation; the CpG annotation; the percentage of enhancer; the difference signal between
schizophrenia and controls in NeuN-positive (NeuN+) and NeuN-negative (NeuN-) nuclei.

Figure 4. Transcriptional regulatory processes in the frontal cortex of schizophrenia subjects and controls. (A) Heatmap of z-score Personalized
PageRank (PPR) for top 10 significantly differentially expressed transcription factors (TFs) (FDR <0.05) ranked by absolute change in PPR for NeuN-
positive (NeuN+) (upper panel) and NeuN-negative (NeuN-) (lower panel) schizophrenia vs control nuclei samples. (B) Overrepresented pathway analysis
(FDR <0.05) for 203 downstream regulatees common to the top four schizophrenia vs control NeuN+-specific transcription factors (TFs) (ZNF333, SOX2,

Figure 4 continued
ZEB1, and RBPJ). (C) Overrepresented pathway analysis (FDR <0.05) for 225 downstream regulatees common to the top four schizophrenia vs control
NeuN--specific TFs (FOS, BCL6, IRF1, and KLF15).
Alterations in antipsychotic-free but not in antipsychotic-treated

schizophrenics
Using preclinical models, it has been suggested that chronic antipsychotic drug administration leads
to long-lasting changes in frontal gene expression and chromatin organization (Kurita et al., 2012;
de la Fuente Revenga et al., 2018), but the epigenomic consequences of antipsychotic treatment
in postmortem human brain samples remain largely unexplored. To validate the separation between
the antipsychotic-free (AF) schizophrenia and antipsychotic-treated (AT) schizophrenia groups, we first
utilized a dimension reduction algorithm – uniform manifold approximation and projection (UMAP)
– to visualize the clustering of each sample with TMM normalized binding affinity matrices or gene
expression files from MOWChIP-seq and RNA-seq, respectively (Figure 5—figure supplement 1).
The separation between AF-schizophrenia and AT-schizophrenia groups is clearly visible at enhancer
and promoter regions for both NeuN+ and NeuN- nuclei.
To further determine the functional relevance of antipsychotic treatment, we aimed to identify the
biological pathways, TFs, or gene expressions dysregulated in the AF-schizophrenia group that were
also reversed to control levels in the AT-schizophrenia group as compared to individually matched
controls. Hence, these represent schizophrenia-associated molecular alterations that are reversed
upon antipsychotic treatment. We calculated the average pairwise difference in PageRank in NeuN+
nuclei from the AF-schizophrenia/control pair cohort. In conjunction with this, we also calculated
the average pairwise difference in PageRank in NeuN+ nuclei from the AT-schizophrenia/control pair
cohort. We then identified those TFs with a difference in these two values greater than 0.5 (Figure 5A;
Supplementary file 13). However, when these TFs were further filtered based on a significance cut-off
of FDR <0.05, no significant TFs were identified to be simultaneously changed in the AF-schizophrenia/
control group and not changed in the AT-schizophrenia/control group (Figure 5C; Supplementary file
13). In parallel, AF-schizophrenia/control DEGs were integrated with genes from the AT-schizophrenia/
control cohort with no case/control differences in expression resulting in a list of 116 cohort-specific
DEGs (Figure 5C; Supplementary file 13). Functional enrichment analysis of these genes resulted in
pathways involved in glutamatergic neurotransmission including ‘Activation of AMPK downstream of
NMDARs’ (FDR 3.66×10–3) (Figure 5G; Supplementary file 13). Structural and functional modifica-
tions of dendritic spines are central to brain development and plasticity (Spruston, 2008). Studies from
postmortem brains of subjects with neurodevelopmental disorders including schizophrenia demon-
strate altered density and morphology of dendritic spines, particularly in the frontal cortex (Glantz
and Lewis, 2000; Black et al., 2004). IQGAP scaffold proteins facilitate the formation of complexes
that regulate cytoskeletal dynamics including microtubules (Cao et al., 2015b). Interestingly, another
significant pathway restored in the AT-schizophrenia group was ‘Rho GTPases activate IQGAPs’ (FDR
3.66×10–3) (Figure 5G; Supplementary file 13). The importance of this pathway was validated by
the analysis of the clusters from the EpiSig pipeline. Thus, taking the top three clusters ranked for
enrichment in H3K27ac and mapping their differential peaks to genes resulted in 166 genes enriched
in pathways including ‘Adherens junctions interactions’ (p-value 1.22×10–4) (Figure 5—figure supple-
ment 2; Supplementary file 14).
Autophagy has been suggested to play an important role in the pathophysiology of schizophrenia
and antipsychotics are known to modulate the process (Merenlender-Wagner et al., 2015). Notably,
the pathways ‘Aggrephagy’ (FDR 3.66×10–3) and ‘Macroautophagy’ (FDR 7.71×10–3) were significantly
enriched (Figure 5G; Supplementary file 13). Expression of the ‘Macroautophagy’ genes AMBRA1,
PRKAB1, TUBA1A, TUBB2A, and TUBA4A was restored in the AT-schizophrenia group (Figure 5I;
Supplementary file 13). Ubiquitin B (UBB) expression has been previously identified as a strong
correlate of schizophrenia symptoms (Rubio et al., 2013). We show a 1.96-fold decrease specific to
AF-schizophrenics compared to controls (p-value =4.0×10–2) (Supplementary file 13).
Glial cells modulate and act as effectors in neurodevelopment through a wide range of neuronal-
glial cell interactions. Using the same process as above, we identified two driver TFs with a signif-
icant change in PPR between case and control for the AF-schizophrenia cohort and no significant

Figure 5. Epigenomic alterations affected by antipsychotic treatment. (A) Scatter plot of average pairwise change in Personalized PageRank (PPR)
(PPRschizophrenia – PPRcontrol) for antipsychotic-free (AF) vs antipsychotic-treated (AT) NeuN-positive (NeuN+) cohorts. Orange regions show cohort (AF
– AT)<0.5 (i.e. alterations recovered by antipsychotic treatment), whereas beige regions show cohort (AT – AF)>0.5 (i.e. alterations consequence of
antipsychotic treatment). Transcription factors (TFs) FDR <0.05 highlighted in red. (B) Scatter plot of average pairwise change in PPR (PPRschizophrenia –

Figure 5 continued
PPRcontrol) for AF vs AT NeuN- cohorts. Dark blue regions show cohort (AF – AT)<0.5 (i.e. alterations recovered by antipsychotic treatment), whereas
cyan regions show cohort (AT – AF)>0.5 (i.e. alterations consequence of antipsychotic treatment). TFs FDR <0.05 highlighted in red. (C) Number of
differentially expressed gene (DEG) regulatees by TFs, and number of DEGs in NeuN+ nuclei from AF-schizophrenia/control pairs. (D) Number of DEG
regulatees by TFs, and number of DEGs in NeuN- nuclei from AF-schizophrenia/control pairs. (E) Number of DEG regulatees by TFs, and number of
DEGs in NeuN+ nuclei from AT-schizophrenia/control pairs. (F) Number of DEG regulatees by TFs, and number of DEGs in NeuN- nuclei from AT-
schizophrenia/control pairs. (G) Functional enrichment analysis of union of genes from AF-schizophrenia/control pairs in NeuN+ nuclei. (H) Functional
enrichment analysis of union of genes from AF-schizophrenia/control pairs in NeuN- nuclei. (I) Pairwise expression difference (schizophrenia – control)
of an exemplar AF-schizophrenia/control cohort DEG (TUBB2A) across all 29 schizophrenia-control pairs in NeuN+ nuclei. (J) H3K27ac tracks for
PDK1 (member of the 84 gene set in E) in NeuN+ nuclei. Box highlighting the FOXO1 DNA-binding motif in promoter at position chr2: 172,555,706–
172,555,718 (GRCh38). Two exemplar AT-schizophrenia/control cohort pairs showing differential H3K27ac peak intensity around motif locus and an
example AF-schizophrenia/control cohort patient pair with no difference.
Figure supplement 1. Uniform manifold approximation and projection (UMAP) visualization of the feature matrix of enhancers, promoters, and RNA
among antipsychotic-free (AF)-schizophrenia, antipsychotic-treated (AT)-schizophrenia, and control subjects.
Figure supplement 2. Differential peak enrichment analysis for antipsychotic-free NeuN-positive (NeuN+) nuclei in EpiSig clusters.
Figure supplement 3. Differential peak enrichment analysis for antipsychotic-treated NeuN-positive (NeuN+) nuclei in EpiSig clusters.
Figure supplement 4. Differential peak enrichment analysis for antipsychotic-treated NeuN-negative (NeuN-) nuclei in EpiSig clusters.
difference between case and control in the AT-schizophrenia group, SOX11 (FDR 1.73×10–2) and MGA
(FDR 9.83×10–3) (Figure 5B; Supplementary file 13). 77 downstream DEG regulatees of these two
TFs were identified in the AF cohort showing significant regulatory case/control change (Figure 5D;
Supplementary file 13). In parallel, 21 cohort-specific DEGs were identified as AF-schizophrenia/
control DEG and having no significant difference in expression in the AT-schizophrenia/control cohort
(Figure 5D; Supplementary file 13). Functional enrichment analysis of the union of 153 genes
included ‘Post NMDA receptor activation events’ (FDR 3.65×10–2), and ‘Long-term potentiation’ (FDR
1.95×10–2) (Figure 5H; Supplementary file 13). EpiSig’s analysis did not show NeuN- alterations in
the AF-schizophrenia cohort.
We also performed the differential analysis with demographic and technical covariates regressed
out on AF-schizophrenia/control and AT-schizophrenia/control cohorts. In NeuN+ nuclei, the results
revealed 2,069 and 574 differential enhancers and promoters, respectively, and 166 DEGs between
AF-schizophrenia and their controls (Figure 6A; Supplementary file 15), while 3,658, 36, and 1,273
differential enhancers, promoters, and DEGs were discovered between AT-schizophrenia and controls
(Figure 6B; Supplementary file 15). In NeuN- nuclei, we identified 891, 19, and 128 differential peaks/
genes between AF-schizophrenia and controls (Figure 6A; Supplementary file 15); 2,651, 775, 776
differential peaks/genes between AT-schizophrenia and controls, in enhancers, promoters, and DEGs,
respectively (Figure 6B; Supplementary file 15). More differential enhances/promoters and genes were
detected between AT-schizophrenia and their matched controls than those between AF-schizophrenia
and their controls with the exception in neuronal promoters (Figure 6A and B; Supplementary file 15).
Similar to our TF analyses (Figure 5), we also identified the genes altered in the AF-schizophrenia/
control group but not in the AT-schizophrenia/control group using differential analyses of enhancers,
promoters, or gene expression. It should be noted that in the differential analyses here, the schizo-
phrenia subjects (whether AF or AT) and their controls were compared at the cohort level, while
matched schizophrenia/control pairs were examined individually in the TF-based analysis. At the
epigenomic level, in NeuN+ nuclei, we identified 687 and 549 genes changed in the AF- but not in
AT-schizophrenics by examining differential enhancers and promoters, respectively (Supplementary
file 16). These genes were linked to epigenomic features restored to their basal level after treat-
ment. In NeuN- nuclei, there were 270 and 17 recovered genes linked with differential enhancers and
promoters, respectively. At the transcriptomic level, 145 DEGs in NeuN+ nuclei and 109 in NeuN-
nuclei were discovered in AF- schizophrenia/control comparison but not in the AT- schizophrenia/
control differential analysis.

Figure 6. Effect of antipsychotic treatment on differential enhancers/promoters and differentially expressed genes (DEGs) in NeuN-positive (NeuN+)
and NeuN-negative (NeuN-) nuclei from the frontal cortex of schizophrenia subjects and controls. (A) Differential enhancer/promoter peaks and DEGs
were obtained by comparing antipsychotic-free (AF)-schizophrenics and individually matched controls. (B) Differential enhancer/promoter peaks and
DEGs obtained by comparing antipsychotic-treated (AT)-schizophrenics and individually matched controls.
Alterations in antipsychotic-treated but not in antipsychotic-free

schizophrenics
We next sorted to characterize those TFs that exhibit regulatory alterations in the AT-schizophrenia/
control cohort but not in the AF-schizophrenia/control cohort. Our goal was to identify modifications
in pathways that represent a consequence of antipsychotic medication rather than an epigenetic mark
of schizophrenia in the postmortem human brain (in other words, unwanted side effects caused by
antipsychotic treatment). In the same way as above, we identified those TFs with a change in the
AT-schizophrenia/control group but not in the AF-schizophrenia/control group for the NeuN+ nuclei
(Figure 5A; Supplementary file 13). Further filtering of these TFs based on a significance cut-off of
FDR <0.05 leads to the identification of FOXO1 (FDR 4.89×10–2).
We identified dysregulated AT-schizophrenia/control DEG regulatees of these TFs in NeuN+ nuclei
via analysis of differential edge weights thus obtaining 84 genes (Figure 5E; Supplementary file 13).
AT-treated/control DEGs were intersected with genes from the AF-schizophrenia/control cohort with
no case/control differences in expression resulting in a list of 41 cohort-specific DEGs (Figure 5E;
Supplementary file 13). Pathway analysis on the union of genes yields the pathway ‘Regulation
of p53 activity through phosphorylation’ (FDR 1.13×10–2) (Supplementary file 13), including the
FOXO1 AT-schizophrenia/control cohort DEG regulatees CCNA1, BLM, TP53RK, and RBBP8, and
the AT-schizophrenia/control cohort-specific DEGs PRKAA1 and TAF15 (Supplementary file 13). The
p53 regulatory gene PDK1 was also identified as a FOXO1 AT-schizophrenia/control DEG regulatee
(Figure 5J; Supplementary file 13). p53 is one of the most critical pro-apoptotic genes, and anti-
psychotics are known to produce complex effects including the activation of both proapoptotic and
antiapoptotic signaling pathways (Aylon and Oren, 2007). Our data showed that all genes involved

in the regulation of p53 presented a significantly lower expression in AT-schizophrenics compared to

controls, suggestive of a repressive role for FOXO1 for its five regulatees, as FOXO1 had higher PPR
in treated schizophrenics (Supplementary file 13) and was also 2.38-fold more highly expressed in
schizophrenics for the treated-cohort (p-value = 1.07×10–2) (Supplementary file 13). Analysis of the
H3K4me3 enriched clusters from the EpiSig pipeline for the AT-schizophrenia/control cohort corrob-
orated alterations in pathways related to p53 (Endo et al., 2008; Figure 5—figure supplement 3;
Supplementary file 17).
In NeuN- nuclei, no TFs showed a significant difference in the AT-schizophrenia/control cohort but
not in the AF-schizophrenia/control cohort (Figure 5F; Supplementary file 13). Furthermore, the 118
genes cohort-specific DEGs identified as AT-schizophrenia/control DEGs and having no significant
difference in expression in the AF-schizophrenia/control cohort were not significantly enriched in any
signaling pathway. Analysis of the clusters from the EpiSig pipeline remarked the importance of the
RHO GTPase pathway on the regulatory alterations observed in AT-schizophrenia subjects (Figure 5—
figure supplement 4; Supplementary file 18).
We also used differential analyses of enhancers, promoters, and expression to discover the genes
altered in the AT- schizophrenia/control group but not in the AF- schizophrenia/control group. In
NeuN+ nuclei, we found 1,591 and 28 treatment-altered genes linked with differential enhancers and
promoters, respectively (Supplementary file 16). In NeuN- nuclei, we identified 1,351 and 718 altered
genes linked with differential enhancers and promoters, respectively (Supplementary file 16). At
the transcriptomic level, 1,252 DEGs in NeuN+ nuclei and 757 in NeuN- nuclei were discovered in
AT-schizophrenia/control comparison but not in the AF-schizophrenia/control differential analysis.
Age differentially affects antipsychotic-treated schizophrenia subjects

In order to further assess the effect of age on gene expression, we first compared transcriptomes
of subjects with schizophrenia and the controls to evaluate how these changes correlated with age.
Within NeuN+ nuclei in the control group, we identified 742 genes that were significantly correlated
with age – with most of them (573, or 77.2%) showing decreased expression in older control subjects
(Figure 7A; Supplementary file 19). These included APOL2, which has been involved in epigenetic
aging (Luo et al., 2020). The opposite, however, was observed in NeuN+ nuclei from schizophrenia
subjects with 18 out of 622 (2.8%) in AF-schizophrenia presenting a negative correlation with age, an
effect that was partly reversed in the AT-schizophrenia cohort (85 out of 242 or 35.1%) (Figure 7A;
Supplementary file 19).
Our data also demonstrate that within the NeuN- nuclei genes correlated with age (1,031, 389,
and 351 in controls, AF-schizophrenia and AT-schizophrenia, respectively), approximately half (491
or 47.6%) were positively correlated with age in the control group whereas a much higher fraction of
genes showed increased expression with age in the schizophrenia group, particularly in the AF-schizo-
phrenia cohort (382 or 98.2% in AF-schizophrenia, and 276 or 78.6% in AT-schizophrenia) (Figure 7B;
Supplementary file 19). These results suggest that age differentially affects gene expression in the
frontal cortex of AF-schizophrenia vs AT-schizophrenia subjects as compared to age-matched controls.
Importantly, this was further confirmed by the functional integration of epigenomic and transcriptomic
data and the evaluation of how these alterations correlated with age.
Thus, we evaluated pairwise changes in expression between schizophrenia subjects and their age-
matched controls, and identified 206 and 310 genes with an absolute Pearson’s correlation of ≥0.50
in NeuN+ nuclei from the AF-schizophrenia/control and AT-schizophrenia/control cohorts, respectively
(Figure 7C and D; Supplementary file 19). We also found enriched biological processes associated
with age, including ‘Regulation of protein kinase activity’ (p-value 6.69×10–7) in the AT-schizophrenia/
control group (Figure 7E; Supplementary file 19). Within this gene set, the difference between
AT-schizophrenia subjects and control pairs correlated either positively (WNK1) or negatively (SFRP2)
with age (Figure 7F and G; Supplementary file 19). Evaluation of pairwise changes in PPR identified
48 TFs with high correlations to age in NeuN+ nuclei from AT-schizophrenia/control cohorts (Figure 7D;
Supplementary file 19), whereas this alteration was not observed in the AF-schizophrenia/control
group (Figure 7C). Pathway analysis of the NeuN+ TFs affected by age in the AT-schizophrenia/control
cohort led to the top pathway ‘NGF-simulated transcription’ (p-value 8.04×10–8), including the TFs
EGR2 and ATF2 (Supplementary file 19). Hallucinations and delusions typically attenuate with aging
(Davidson et al., 1995), which is consistent with the lower PPR difference for EGR2 – a preclinical

Figure 7. Epigenomic effect of age on treated schizophrenia subjects. (A) Violin plots for Pearson’s R correlation coefficients of age vs expression f 742,
622, and 242 genes from control, antipsychotic-free (AF)-schizophrenia, and antipsychotic-treated (AT)-schizophrenia NeuN-positive (NeuN+) nuclei,
respectively. (B) Violin plots for Pearson’s R correlation coefficients of age vs expression for 1031, 389, and 351 genes from control, AF-schizophrenia, and
AT-schizophrenia NeuN-negative (NeuN-) nuclei, respectively. (C) Number of pairwise transcription factor (TF) Personalized PageRank (PPR) and pairwise

Figure 7 continued
gene expression differences correlated with age in NeuN+ nuclei from AF-schizophrenia/control pairs. (D) Number of pairwise TF PPR and pairwise
gene expression differences correlated with age in NeuN+ nuclei from AT-schizophrenia/control pairs. (E) Heatmap for the 14 age positively-correlated
(schizophrenia – control increase with age) and 12 age negatively-correlated (schizophrenia – control decrease with age) genes of the significant
GO term ‘Regulation of kinase activity’ from the AT-schizophrenia/control NeuN+ cohort. (F) Example gene, WNK1 pairwise expression difference
(schizophrenia – control) vs age (Pearson’s R=0.73; p-value = 0.003). (G) Example gene, SFRP2 pairwise expression difference (schizophrenia – control)
vs age (Pearson’s R=–0.70; p-value = 0.005). (H) Example TF, EGR2 pairwise PPR difference (schizophrenia – control) vs age (Pearson’s R=0.69; p-value
= 0.0003) (I) Number of pairwise TF PPR and pairwise gene expression differences correlated with age in NeuN- nuclei from AF-schizophrenia/control
pairs. (J) Number of pairwise TF PPR and pairwise gene expression differences correlated with age in NeuN- nuclei from AT-schizophrenia/control pairs.
(K) Heatmap for the 5 age positively-correlated (schizophrenia – control increase with age) genes of the significant GO term ‘Beta-catenin independent
WNT signaling’ from the AT-schizophrenia/control NeuN- cohort.
marker of psychosis-like behavior (González-Maeso et al., 2007) – that we observed in older subjects
(Figure 7H; Supplementary file 19).
In NeuN- nuclei, 147 and 88 genes were identified with AF-schizophrenia/control and AT-schizo-
phrenia/control expression difference vs age correlations of ≥0.60, respectively (Figure 7I and J;
Supplementary file 19). Enriched pathways in the AT-schizophrenia/control group included: ‘Degra-
dation of DVL’ (p-value 4.04×10–5) and ‘Beta-catenin independent WNT signaling’ (p-value 5.06×10–4)
(Figure 7K; Supplementary file 19). Since dysfunctional WNT signaling is associated with several
CNS disorders including Alzheimer’s (Wan et al., 2014), together, these data also suggest that this
positive correlation between NeuN- gene differences in AT-schizophrenia subjects/control pairs and
age (Figure 7K; Supplementary file 19) may be responsible for some of the negative effects of
antipsychotic treatment on cognitive processes. We also identified 11 and 53 TFs correlated with
age in the AF-schizophrenia/control and AT-schizophrenia/control cohorts, respectively (Figure 7I and
J; Supplementary file 19). However, as in NeuN+ nuclei, the effect of age became more evident in
the AT-schizophrenia/control group with age-related adaptations in NeuN- TF-affected pathways that
included ‘Signaling by NOTCH’ (p-value 3.2×10–4) (Supplementary file 19).
Discussion
Understanding the molecular determinants involved in schizophrenia is critical for devising new treat-
ment strategies and the discovery of the pathogenic mechanisms underlying this psychiatric condi-
tion. In this study, we combined low-input epigenomic and transcriptomic analysis to define how
gene expression and TF regulation vary in schizophrenia subjects relative to controls and in response
to antipsychotic treatment and aging. Our data provide evidence suggesting alterations in cova-
lent histone modifications at different gene regions previously associated with schizophrenia risk,
as well as additional genes involved in pathways related to immunological and neurodevelopmental
processes. Whereas previous studies with postmortem human brain samples compared using indirect
methods differences in chromatin accessibility between schizophrenia subjects and controls (Bryois
et al., 2018), here we provide epigenomic signatures that distinguish between those observed in
AF-schizophrenia subjects as well as alterations that denote previous treatment with antipsychotic
medications.
We conducted a pairwise comparison between schizophrenia and matched controls, which is crucial
to tease out treatment- or age-associated effects. A powerful feature of the Taiji framework is to allow
the analysis of individual samples (Zhang et al., 2019). This integrative analysis of transcriptomic and
epigenomic data at the systems level uncovered key regulators and important pathways based on
their global importance in the genetic networks. We found that transcriptional mechanisms via novel
pathways that had not been previously associated with schizophrenia show alterations in AF-schizo-
phrenia subjects, and that these schizophrenia-linked pathways were statistically unaffected in the
AT-schizophrenia group, consistent with a potential role of these epigenomic signatures in the clinical
efficacy of antipsychotics. These changes appear to impact glutamatergic neurotransmission, IQGAP
scaffold, autophagy, and ubiquitin B expression in neurons; and post NMDA receptor activation and
long-term potentiation in glial cells. Additionally, our data highlight processes related to key pathways
that may represent a consequence of antipsychotic medication, rather than a reversal of the molecular
alterations observed in AF-schizophrenia subjects. These pathways suggest the existence of compen-
satory perturbations that emerge in response to repeated antipsychotic drug administration and

ultimately restrain their therapeutic effects (Kurita et al., 2012; Ibi et al., 2017). Among these, alter-
ations in p53 activity were apparent as a consequence of antipsychotic treatment. Based on our ability
to individually match schizophrenia and control pairs by age, we also revealed the intriguing observa-
tion that the effect of age on TF regulation of gene expression was significantly more pronounced in
AT-schizophrenia subjects as compared to AF-schizophrenia subjects and controls.
Related to the effect of antipsychotic treatment, frontal cortex samples of schizophrenia subjects
were divided into AF and AT based on postmortem toxicological analysis in both blood and when
possible brain samples, which provides information about a longer retrospective drug-free period
due to the high liposolubility of antipsychotic medications (Voicu and Radulescu, 2009). However, we
cannot fully exclude the possibility of previous exposure to antipsychotic medications in the AF-schizo-
phrenia group, and hence that the epigenetic alterations observed exclusively in the AF-schizophrenia
group are a consequence of a potential period of decompensation, which typically occurs following
voluntary treatment discontinuation (Liu-Seifert et al., 2005). It is also worth noting that our find-
ings were established by examining the average characteristics of entire NeuN+ and NeuN- fractions.
Further studies of individual neuronal and glial cell subtypes may yield additional information on the
role of cell-type-subpopulations (Lau et al., 2020; Nagy et al., 2020).
Conclusion
Our ChIP-seq/RNA-seq study in postmortem brain samples from schizophrenia subjects and controls
suggests cell-type specific epigenomic differences in individuals with schizophrenia, as well as cellular
alterations in signaling pathways potentially involved in either the elimination of schizophrenia-related
epigenomic alterations upon antipsychotic drug treatment or the antipsychotic-dependent modula-
tion of alternative epigenetic pathways previously unaffected in the untreated schizophrenia cohort.
Building on our data, future research could test the causal role of specific molecular pathways impli-
cated in schizophrenia pathophysiology, as well as the therapeutic versus compensatory or negative
side epigenomic outcomes induced by chronic treatment with antipsychotic medications.
Materials and methods

Post-mortem human brain tissue samples
Human brains were obtained during autopsies performed at the Basque Institute of Legal Medi-
cine, Bilbao, Spain. The study was developed in compliance with policies of research and ethical
review boards for post-mortem brain studies (Arias-Diaz et al., 2013). The Institutional Review Board
(IRB) of the University of the Basque Country determined that approval was not required for this
research in postmortem samples (M10/2018/283). According to legal requirements, samples were
obtained by opting-out policy, absence of compensation for tissue donation and coded under revers-
ible anonymization. Deaths were subjected to retrospective searching for previous medical diagnosis
and treatment using examiner’s information and records of hospitals and mental health centers. After
searching for antemortem information was fulfilled, 29 subjects (Caucasian) who had met criteria of
schizophrenia according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) (Amer-
ican Psychiatric Association, 1994) were selected. A toxicological screening for antipsychotics,
other drugs, and ethanol was performed on blood samples collected at the time of death and, when
possible, postmortem brain samples. The toxicological assays were performed at the National Insti-
tute of Toxicology, Madrid, Spain, using a variety of standard procedures including radioimmunoassay,
enzymatic immunoassay, high-performance liquid chromatography, and gas chromatography-mass
spectrometry. Controls (Caucasian) for the present study were chosen among the collected brains on
the basis, whenever possible, of the following cumulative criteria: (i) negative medical information on
the presence of neuropsychiatric disorders or drug abuse; (ii) appropriate sex, age, postmortem delay
(time between death and autopsy), and freezing storage time to match each subject in the schizo-
phrenia group; (iii) sudden and unexpected death (motor vehicle accidents); and (iv) toxicological
screening for psychotropic drugs with negative results except for ethanol. Specimens of frontal cortex
(Brodmann area 9) were dissected at autopsy (0.5–1 g tissue) on an ice-cooled surface and immedi-
ately stored at –80 °C until use. The schizophrenia subjects were divided into antipsychotic-free and
antipsychotic-treated according to the presence or absence of antipsychotics in blood samples at
the time of death. The definitive pairs of antipsychotic-free schizophrenics and respective matched

controls are shown in Supplementary file 1, and the definitive pairs of atypical antipsychotic-treated
schizophrenics and respective matched controls are shown in Supplementary file 2. Presence or
absence of antipsychotic medications was confirmed by toxicological analysis in postmortem brain
samples of a selected group of schizophrenia subjects and controls (Supplementary file 20). Pairs of
schizophrenia and matched controls were processed simultaneously and under the same experimental
conditions. Tissue pH values were within a relatively narrow range (control subjects: 6.7±0.08; schizo-
phrenic subjects: 6.6±0.06). Brain samples were also assayed for RIN (RNA integrity number) values
using the Agilent 2100 Bioanalyzer (Applied Biosystems) – control subjects: 7.87±0.21; schizophrenic
subjects: 7.61±0.32.
Nuclei isolation and sorting via FACS

Nuclei isolation from frozen tissues (never fixed) of postmortem human brain samples was conducted
using a published protocol (Lake et al., 2016) with some modifications. Frontal cortex samples from
schizophrenic individuals and individually matched controls were always processed in the same batch.
Briefly, all steps were conducted on ice, and all centrifugation was conducted at 4 °C. One piece of
brain tissue (~300 mg) was placed in 3 ml of ice-cold nuclei extraction buffer (NEB) [0.32 M sucrose,
5 mM CaCl2, 3 mM Mg(Ac)2, 0.1 mM EDTA, 10 mM Tris-HCl, and 0.1%(v/v) Triton X-100] with freshly
added 30 μl of protease inhibitor cocktail (PIC, Sigma-Aldrich), 3 μl of 100 mM phenylmethylsulfonyl
fluoride (PMSF, Sigma-Aldrich) in isopropyl alcohol, 3 μl of 1 M dithiothreitol (DTT, Sigma-Aldrich),
and 4.5 μl of recombinant ribonuclease (RNase) inhibitor (2313 A,Takara Bio). The tissue was homoge-
nized in tissue grinder (D9063, Sigma-Aldrich). The homogenate was filtered with a 40 μm cell strainer
(22-363-547, Thermo Fisher Scientific) and collected in a 15 ml centrifuge tube. The cell suspension
was centrifuged at 1000 RCF at 4 °C for 10 min. The supernatant was discarded, and the pellet was
resuspended in 0.5 ml of ice-cold NEB with freshly added 5 μl of PIC, 0.5 μl of PMSF, 0.5 μl of DTT,
and 0.75 μl of RNase inhibitor. 500 μl of the sample was mixed with 750 μl of 50%(w/v) iodixanol (a
mixture of 4 ml of OptiPrepTM gradient (Sigma-Aldrich) and 0.8 ml of diluent [150 mM KCl, 30 mM
MgCl2, and 120 mM Tris-HCl]). The mixture was centrifuged at 10,000 RCF at 4 °C for 20 min. Then,
the supernatant was removed and 300 μl of 2%(w/v) normal goat serum (50062Z, Life technologies)
in Dulbecco’s PBS (DPBS, Life technologies) was added to resuspended the nuclei pellet. To label and
separate NeuN+ and NeuN- fractions, 6 μl of 2 ng/ml anti-NeuN antibody conjugated with Alexa 488
(MAB377X, Millipore) in DPBS was added into the nuclei suspension. The suspension was mixed well
and incubated at 4 °C for 1 hr on a rotator mixer (Labnet). After incubation, the sample was sorted
into NeuN+ and NeuN- populations using a BD FACSARIATM Flow Cytometer (BD Biosciences). 400 μl
of sorted nuclei suspension (NeuN+ or NeuN-), containing ~50,000 nuclei (for conducting ChIP-seq
and input libraries), was added into 600 μl of ice-cold PBS. 200 μl of 1.8 M sucrose solution, 5 μl of
1 M CaCl2, and 3 μl of 1 M Mg(Ac)2 were added into the mixture. The solution was mixed well and
incubated on ice for 15 min. Then, the sample was centrifuged at 1800 RCF at 4 °C for 15 min. The
supernatant was discarded and the pellet was resuspended in 110 μl of PBS with freshly added 1.1 μl
of PIC and 1.1 μl of PMSF and stored on ice until use.
Construction of ChIP-seq libraries

Chromatin fragments were prepared by using micrococcal nuclease (MNase) to digest sorted and
concentrated nuclei (NeuN+/NeuN-) following a published protocol (Zhu et al., 2019). 54 µl of chro-
matin fragments (from 10,000 nuclei) was used in each ChIP assay for producing two replicate libraries.
Chromatin immunoprecipitation was carried out using multiplexed MOWChIP assay (Zhu et al., 2019)
with anti-H3K4me3 (39159, Active Motif) and anti-H3K27ac (39135, Active Motif) antibody. ChIP-seq
libraries were prepared using Accel-NGS 2 S Plus DNA Library kit (Swift Biosciences) from the purified
immunoprecipitated DNA. The library preparation was conducted without knowledge of the brain
sample or the type of histone mark. Minor modification was made to the manufacturer’s procedures
as detailed below. In the amplification step, instead of adding 10 µl of low EDTA TE buffer into each
reactoin, we added the mixture of 7.5 µl of low EDTA TE buffer and 2.5 µl of 20 X Evagreen dye to
monitor and quantify PCR amplification. The reaction was stopped when the sample’s fluorescence
intensity increased by 3000 relative fluorescence units (RFU). Then, 50 µl of the mixture after PCR
amplification was transferred into an Eppendorf tube and mixed with 37.5 µl of SPRI select beads.

After 5 min incubation at room temperature, the beads went through a cleanup procedure with 80%
ethanol. In the end, the DNA library was eluted from the beads into 7 µl of low EDTA TE buffer.
Construction of RNA-seq libraries

100 μl of sorted nuclei suspension (NeuN+ or NeuN-) from brain tissue, containing ~12,000 nuclei for
producing two replicate libraries, was used for RNA extraction by using the RNeasy Mini Kit (74104,
Qiagen) and RNase-Free DNase Set (79254, Qiagen), following the manufacturer’s instruction. Half
of the extracted mRNA (from 6000 nuclei) in 30 µl volume was concentrated by ethanol precipitation
and resuspended in 4.6 μl of RNase-free water. mRNA-seq libraries were prepared using Smart-seq2
(Picelli et al., 2013) and a Nextera XT DNA Library Preparation kit (FC-131–1024, Illumina) following
the protocol and the manufacturer’s instructions with minor modification. ~2 ng of mRNA (in 4.6 µl
of water) was mixed with 2 μl of 100 mM oligo-dT primer and 2 μl of 10 mM dNTP mix. After being
denatured at 72 °C for 3 min, the mRNA solution was immediately placed on ice. Then, 11.4 μl of
reverse transcript mix [1 μl of SuperScript II reverse transcriptase (200 U/ml), 0.5 μl of RNAse inhibitor
(40 U/ml), 4 μl of Superscript II first-strand buffer, 1 μl of DTT (100 mM), 4 μl of 5 M Betaine, 0.12 μl
of 1 M MgCl2, 0.2 μl of TSO (100 mM), 0.58 μl of nuclease-free water] was mixed with the mRNA
solution and the mixture was incubated at 42 °C for 90 min, followed by 10 cycles of (50 °C for 2 min,
42 °C for 2 min). The reaction was finally inactivated at 70 °C for 15 min. 20 μl of first-strand mixture
was then mixed with 25 μl of KAPA HiFi HotStart ReadyMix, 0.5 μl of (100 mM) IS PCR primers,
0.5 μl of Evagreen dye, and 4 μl of nuclease-free water. Generated complementary DNA (cDNA) was
amplified by incubated at 98 °C for 1 min, followed by 9–11 cycles of (98 °C 15 s, 67 °C 30 s, 72 °C
6 min). After PCR amplification, 50 μl of PCR mix was purified by using 50 μl of SPRIselect beads.
~600 pg of purified cDNA was used for Nextera XT library preparation. ChIP-seq and RNA-seq library
fragment size were measured by using high sensitivity DNA analysis kit (5067–4626, Agilent) on a
TapeStation system (2200, Agilent). After this, 18–22 ChIP-seq and RNA-seq libraries were randomly
pooled together. Around 15 and 11 million reads were allocated to each ChIP-seq and RNA-seq
library, respectively. The concentration of each library was examined by a KAPA library quantification
kit (KK4809, Kapa Biosystems), and then the quantified libraries were pooled at 10 nM. The libraries
were sequenced by Illumina HiSeq 4000 with single-end 50-nt read.
ChIP-seq data processing

Raw ChIP-seq reads and input data, were mapped to human genome (GRCh38) using Bowtie2 (2.2.5).
Peaks were called using MACS2 (2.2.7.1) using a q-value cutoff of 0.05 for the narrow marks (H3K4me3
and H3K27ac).
RNA-seq data processing

The human genome (GRCh38) and comprehensive gene annotation were obtained from GENCODE
(v29). Quality control of RNA-seq reads including per sequence GC and adapter content was assessed
with FastQC. Reads were mapped with STAR (2.7.0 f) with soft-clipping (average of 73.8% (+/-0.08%)
reads uniquely mapped for neurons and 69.0% (+/-0.99%) reads for glia) and quantified with feature-
Counts (v2.0.1) using the default parameters.
Differential analysis for ChIP-seq data

The peaks were called using MACS2 (Zhang et al., 2008). The peaks with q-value <0.05 were taken
as input for diffBind R package. We first created cell-type-specific consensus peak sets using Diffbind
for neurons and glia separately. Using the function of dba.peakset in diffbind (hg38_blacklist_remove,
consensus = DBA_REPLICATE, minOverlap = 2), we detected the ‘high-confidence’ peaks if they were
identified in both of the technical replicates of the sample (n=58) in either schizophrenia or control
groups and then the ‘high-confidence’ peak sets from each sample of the two groups were combined
into a master set of consensus peaks for analysis. The raw read counts were extracted using the
function of dba.count (hg38_blacklist_remove, summits = FALSE, peaks = consensus_peaks,filter = 1,
bScaleControl = TRUE, minCount = 1, score = DBA_SCORE_TMM_MINUS_FULL) in diffBind, and the
peaks with less than 20 reads in over 50% of the samples were removed before differential analysis.
DESeq2 R package was used to perform the differential peaks analysis based on the TMM normal-
ized reads to identify differential peaks between schizophrenia and control cohort (adjusted p-value

<0.05). The p-values were adjusted by performing a standard Bonferroni correction. The following
covariates were regressed out: demographic covariates (age at death, sex, PMD, and diagnosis) and
technical covariates (align rate, unique rate, FRiP, NSC, RSC, the number of identified peaks, and
PBC) by correlating the top 6 principal components with these covariates. We annotated enhancers
(defined as identified H3K27ac peaks that have no overlap regions with promoters) to genes using
published Hi-C data on neurons and glia (Hu et al., 2021) when possible and the rest of the enhancers
were associated with their nearest genes. We annotated H3K4me3 peaks to genes when they over-
lapped with the promoter regions.
ChIP-seq annotation and functional enrichment

GREAT analysis (http://great.standford.edu) was performed on differential peaks using the whole
genome as background and default basal extension from 5 kb upstream to 1 kb downstream of the
TSS. Significantly enriched Gene Ontology biological processes were identified using the Panther
Classification tools using a hypergeometric test.
Differential analysis for RNA-seq data

We analyzed the bulk RNA-seq data of 29 schizophrenia subjects and 29 controls. The initial step
involved filtering out genes with low read counts (less than 20 reads in over 50% of samples). The anal-
ysis then employed a two-step method to estimate the technical and biological noise. The first step
was identifying the top 10 principal components (PCs) of the dataset. Subsequently, the correlation
between each PC and various experimental (alignment rate, unique rate, exon percentage, number
of unique mapped reads) and demographic (sex, age at death, PMD, antemortem diagnosis) factors
was calculated. Covariates with high correlation to the PCs were included in the analysis to minimize
their impact. The analysis was conducted using the 'DESeq2' software package, and genes with a false
discovery rate (FDR) below 0.05 were identified as differentially expressed.
ChIP-qPCR assays
After nuclei extraction, MNase digestion and MOWChIP assays (see above), ChIP DNA was eluted to
10 µl of low EDTA TE buffer. 1 µl of ChIP DNA solution was used for qPCR assays with each primer set.
The following qPCR primer pairs were used:
Figure 2—figure supplement 1A: AGG G AC C TG GAA C AT C TT T G (F); CAT C AT C

CT CAG
AAG G AG T CT G (R)
Figure 2—figure supplement 1B: TGG AGA TAG GTG GAT GTT AAG C (F); CCA TAT TGA
CCC T GG GCT A TT (R)
Figure 2—figure supplement 1C: ATG CCA A TT A
GG C TA T AG ATG C T (F); CTT AAC AGG
GCA C TC T
CA G
TA A T (R)
Figure 2—figure supplement 1D: AAA G AG C AA G CA GGG A CT T (F); GAT G TA A TA A
CG
TGG G AG A GA GG (R)
Figure 2—figure supplement 1E AGG A GT GGA T AC AGG GAG ATT AG (F); TGT G TA T
TC
TGT GTC T GG CTT T (R)
Figure 2—figure supplement 1F: ACC AAC GAA T AC C
CT G CT T T (F); AAG GCC TGG CAA
CCT T AA T
(R)
The following common negative primer set was used in all samples, against which the enrichment
of each positive set was calculated:
GCA G
AA C
CT AGT T
CC TCC T
TC AAC (F); AGT C
AT C
CC T
TC CTA C
AG A
CT GAG A (R)
qPCR primer sets were ordered from IDT, made to lab-ready formulation (100 µM in low EDTA TE
buffer). Ready to use stocks of primer sets were made by combining 10 µl each of both forward and
reverse primers of the same set with 80 µl of low EDTA TE buffer. 10 µl of iQ SYBR Green Supermix,
1.6 µl of primer stock, 1 µl of ChIP DNA, and 7.4 µl of ultrapure water were added to each qPCR well.
Reaction was conducted on a CFX96 real-time PCR machine (Bio-Rad) with C1000 thermal cycler base.
All PCR assays were performed using the following thermal cycling profile: 95 °C for 10 min followed
by 40 cycles of (95 °C for 15 s, 58 °C for 40 s, 72 °C for 30 s). Relative fold enrichment of each positive

primer (P) against the common negative primer (N) was calculated using the following equation:
Enrichment = 2Cq(N) - Cq(P).
Taiji pipeline
Active regulatory elements were first identified via the overlap of high confidence peaks from H3K27ac
with known gene promoter regions (4kbp upstream and 1kbp downstream of the transcription start
sites). The distal H3K27ac peaks were assigned to active promoters using the unsupervised learning
method EpiTensor, and assigned as an enhancer-promoter interaction if one locus overlapped with
the distal peak and the other locus in the pair overlapped with a known promoter. Putative TF binding
motifs were curated from the CIS-BP database (Weirauch et al., 2014). Using FIMO’s algorithm (Grant
et al., 2011). TFs were identified as having binding sites within 150 bp regions centered around
H3K27ac peak summits. 58 unique NeuN+ (29 schizophrenia and 29 control) and 58 unique NeuN- (29
schizophrenia and 29 control) network topologies were thus constructed by forming directed edges
between TF and their regulatees, if the TF had a predicted binding site in the gene’s promoter or
linked enhancer.
Personalized PageRank (PPR)

The PPR algorithm was run to measure the global influence of each node. To initialize the networks,
node weights were initialized separately in each cell-type i, where a gene’s relative expression level is
a z-score transformation of its absolute expression, zi and the node weight for this gene in cell type
i is then given by ezi. Edge weights were determined according to the expression level of the parent
node TF and the pooled H3K27ac ChIP-seq peak intensity (strength of the TF-gene association) as
previously reported (Zhang et al., 2019). The directionality of the topological edges was reversed
and the normalized node weights were then used as the seed vector for the PPR calculation. Post
convergence, edge directionality was re-reversed.
EpiSig analysis
To integrate H3K27ac, H3K4me3, and RNA-seq data from two cell types across the postmortem frontal
cortex samples from schizophrenia subjects and controls, EpiSig was employed (Ai et al., 2018). This
algorithm detects the significant signals from sequencing data in 5 kb bins across the whole genome,
and then clusters the regions based on the similar epigenomic profiles across all samples.
EpiSig differential enrichment analysis

A hypergeometric test was applied to all EpiSig clusters to assess the enrichment of differential
H3K27ac and H3K4me3 peaks and differentially expressed genes. Clusters with FDR <0.05 were
selected and then ranked according to the number of overlapping peaks for each mark. Peaks were
then mapped to genes using GREAT with default settings.
Age correlation analysis

Raw expression, pairwise expression and pairwise TF PPR age correlations were calculated using the
Pearson R correlation. Significance was assessed by calculating p-values for the Pearson R correlations
using the t-distribution with n-2 degrees of freedom for the respective cohort.
Acknowledgements
CL dedicates this paper to his elder brother Lv Wei who passed away in 2023 after a 30 year battle
with schizophrenia. The authors thank the staff members of the Basque Institute of Legal Medicine
for their cooperation in the study. National Institutes of Health R01MH084894 (JG-M), R01MH111940
(JG-M), R01GM143940 (CL), R01GM141096 (CL), R01HG009626 (WW), R01AI50282 (WW), Eusko
Jaurlaritza IT1211-19 (JJM) and IT-1512/22 (LFC), and VCU Presidential Request Fund (JG-M).
Additional information
Competing interests

J Javier Meana: received unrestricted funds from Janssen. Javier González-Maeso: has sponsored
research contracts with Terran Biosciences and Gonogo Solutions. The other authors declare that no
competing interests exist.
Funding
Funder Grant reference number Author
National Institutes of R01MH084894 Javier González-Maeso

Health
National Institutes of R01MH111940 Javier González-Maeso

Health
National Institutes of R01GM143940 Chang Lu

Health
National Institutes of R01HG009626 Wei Wang

Health
National Institutes of R01AI50282 Wei Wang

Health
Virginia Commonwealth VCU Presidential Request Javier González-Maeso

University Fund
National Institutes of R01GM141096 Chang Lu

Health
Eusko Jaurlaritza IT1211-19 J Javier Meana
Eusko Jaurlaritza IT-1512/22 Luis F Callado
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
Author contributions
Bohan Zhu, Richard I Ainsworth, Data curation, Formal analysis, Investigation, Methodology, Writing
– original draft; Zengmiao Wang, Zhengzhi Liu, Formal analysis, Investigation; Salvador Sierra, Investi-
gation, Methodology; Chengyu Deng, Investigation; Luis F Callado, J Javier Meana, Resources, Inves-
tigation; Wei Wang, Chang Lu, Conceptualization, Resources, Data curation, Software, Formal analysis,
Supervision, Funding acquisition, Methodology, Writing – review and editing; Javier González-Maeso,
Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Meth-
odology, Writing – review and editing
Author ORCIDs
Bohan Zhu ‍ ‍http://orcid.org/0009-0003-9823-8630
Richard I Ainsworth ‍ ‍http://orcid.org/0000-0002-3350-5692
Luis F Callado ‍ ‍https://orcid.org/0000-0001-9941-012X
J Javier Meana ‍ ‍https://orcid.org/0000-0002-7913-6714
Wei Wang ‍ ‍http://orcid.org/0000-0003-4377-5060
Chang Lu ‍ ‍http://orcid.org/0000-0003-0181-5888
Javier González-Maeso ‍ ‍https://orcid.org/0000-0003-3105-3204
Ethics
Human brains were obtained during autopsies performed at the Basque Institute of Legal Medi-
cine, Bilbao, Spain. The study was developed in compliance with policies of research and ethical
review boards for post-mortem brain studies (Arias-Diaz et al 2013). The Institutional Review Board
(IRB) of the University of the Basque Country determined that approval was not required for this
research in postmortem samples (M10/2018/283). According to legal requirements, samples were
obtained by opting-out policy, absence of compensation for tissue donation and coded under revers-
ible anonymization.
Peer review material

Reviewer #1 (Public Review): https://doi.org/10.7554/eLife.92393.3.sa1
Author response https://doi.org/10.7554/eLife.92393.3.sa2

Additional files
Supplementary files
• Supplementary file 1. Demographic information of antipsychotic-free (AF)-schizophrenia subjects
and controls.
• Supplementary file 2. Demographic information of antipsychotic-treated (AT)-schizophrenia
subjects and controls.
• Supplementary file 3. Quality control measurements on ChIP-seq datasets.
• Supplementary file 4. Quality control measurements on RNA-seq datasets.
• Supplementary file 5. Transcription frequency in transcripts per kilobase million (TPM) on key
marker genes for neuronal and glial nuclei samples from control and schizophrenia subjects.
• Supplementary file 6. Differential histone modification peaks and differentially expressed genes
(DEGs) obtained by comparing schizophrenia and controls.
• Supplementary file 7. Enrichment of various genome-wide association studies (GWAS) traits in
differential enhancers and promoters.
• Supplementary file 8. Overlap of differentially expressed genes (DEGs) identified in our study and
a previous snRNA-seq study.
• Supplementary file 9. Genes identified via differential analyses of enhancers, promoters, and RNA
by comparing schizophrenia and controls.
• Supplementary file 10. EpiSig analysis in NeuN-positive (NeuN+) nuclei from the frontal cortex of
schizophrenia subjects and controls.
• Supplementary file 11. EpiSig analysis in NeuN-negative (NeuN-) nuclei from the frontal cortex of
schizophrenia subjects and controls.
• Supplementary file 12. Taiji analysis in NeuN-positive (NeuN+) and NeuN-negative (NeuN-) nuclei
from the frontal cortex of schizophrenia subjects and controls.
• Supplementary file 13. Taiji analysis in NeuN-positive (NeuN+)and NeuN-negative (NeuN-) nuclei
from the frontal cortex of antipsychotic-free (AF)-schizophrenia subjects, antipsychotic-treated (AT)-
schizophrenia subjects, and controls.
antipsychotic-free (AF)-schizophrenia subjects and controls.
• Supplementary file 15. Differential H3K27ac/H3K4me3 peaks and DEGs obtained by comparing
antipsychotic-free (AF)-schizophrenia and antipsychotic-treated (AT)-schizophrenia with their
respective matched controls.
• Supplementary file 16. Genes altered in antipsychotic-free (AF-) but not antipsychotic-treated (AT)-
schizophrenics, and those altered in AT- but not AF-schizophrenics. The lists of genes were obtained
by differential analyses of enhancers, promoters, and RNA in AF-schizophrenia, AT-schizophrenia,
and their respective controls.
antipsychotic-treated (AT)-schizophrenia subjects and controls.
• Supplementary file 18. EpiSig analysis in NeuN-negative (NeuN-) nuclei from the frontal cortex of
antipsychotic-treated (AT)-schizophrenia subjects and controls.
• Supplementary file 19. Effect of age on chromatin organization in the frontal cortex of AF-
schizophrenia subjects, AT-schizophrenia subjects, and controls.
• Supplementary file 20. Toxicological analysis in postmortem human brain samples.
• MDAR checklist
Data availability
The raw ChIP-seq and RNA-seq data were deposited in dbGaP under accession number phs002487.
v1.p1. The processed data can be accessed via Gene Expression Omnibus (GEO) under accession
number GSE174407. The code used for the analysis shown in this manuscript was deposited on
GitHub (copy archived at Chang Lu lab, 2023).

The following datasets were generated:
Author(s) Year Dataset title Dataset URL Database and Identifier
Zhu B, Deng C, Lu C 2021 Antipsychotic- http://www.ncbi. NCBI Gene Expression

induced epigenomic nlm.nih.gov/geo/ Omnibus, GSE174407
reorganization in frontal query/acc.cgi?acc=
cortex of individuals with GSE174407
schizophrenia
Zhu B, Ainsworth RI 2023 Epigenomes and https://www.ncbi.nlm. dbGaP, phs002487.v1.p1

Transcriptomes of Brain nih.gov/projects/gap/
Samples of Schizophrenics cgi-bin/study.cgi?
and Controls study_id=phs002487.
v1.p1
The following previously published dataset was used:
Author(s) Year Dataset title Dataset URL Database and Identifier
Girdhar et al 2018 PsychENCODE Knowledge https://doi.org/10. Synapse, 10.7303/

Portal 7303/syn4566010 syn4566010
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Zhu, Ainsworth et al. eLife 2023;12:RP92393. DOI: https://doi.org/10.7554/eLife.92393  1 of 29

Zhu, Ainsworth et al. eLife 2023;12:RP92393. DOI: https://doi.org/10.7554/eLife.92393  2 of 29

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Epigenome and transcriptome profiling in neuronal and non-neuronal

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Alterations in antipsychotic-free but not in antipsychotic-treated

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Alterations in antipsychotic-treated but not in antipsychotic-free

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in the regulation of p53 presented a significantly lower expression in AT-­schizophrenics compared to

Age differentially affects antipsychotic-treated schizophrenia subjects

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Materials and methods

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Nuclei isolation and sorting via FACS

Construction of ChIP-seq libraries

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Construction of RNA-seq libraries

ChIP-seq data processing

RNA-seq data processing

Differential analysis for ChIP-seq data

Zhu, Ainsworth et al. eLife 2023;12:RP92393. DOI: https://doi.org/10.7554/eLife.92393  20 of 29

ChIP-seq annotation and functional enrichment

Differential analysis for RNA-seq data

Figure 2—figure supplement 1A: AGG G ​ AC C​ TG ​GAA C ​ AT C ​ TT T​ G (F); CAT C​ AT C

Zhu, Ainsworth et al. eLife 2023;12:RP92393. DOI: https://doi.org/10.7554/eLife.92393  21 of 29

Personalized PageRank (PPR)

EpiSig differential enrichment analysis

Age correlation analysis

Zhu, Ainsworth et al. eLife 2023;12:RP92393. DOI: https://doi.org/10.7554/eLife.92393  22 of 29

National Institutes of R01MH084894 Javier González-Maeso

National Institutes of R01MH111940 Javier González-Maeso

National Institutes of R01GM143940 Chang Lu

National Institutes of R01HG009626 Wei Wang

National Institutes of R01AI50282 Wei Wang

Virginia Commonwealth VCU Presidential Request Javier González-Maeso

National Institutes of R01GM141096 Chang Lu

Eusko Jaurlaritza IT1211-19 J Javier Meana

Eusko Jaurlaritza IT-1512/22 Luis F Callado

Peer review material

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The following datasets were generated:

Author(s) Year Dataset title Dataset URL Database and Identifier

Zhu B, Deng C, Lu C 2021 Antipsychotic-­ http://www.​ncbi.​ NCBI Gene Expression

Zhu B, Ainsworth RI 2023 Epigenomes and https://www.​ncbi.​nlm.​ dbGaP, phs002487.v1.p1

The following previously published dataset was used:

Author(s) Year Dataset title Dataset URL Database and Identifier

Girdhar et al 2018 PsychENCODE Knowledge https://​doi.​org/​10.​ Synapse, 10.7303/

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in the regulation of p53 presented a significantly lower expression in AT-schizophrenics compared to

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Figure 2—figure supplement 1A: AGG G AC C TG GAA C AT C TT T G (F); CAT C AT C

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